Heart disease is one of the most common causes of death in the world and the most troublesome symptoms. Accordingly, a large number of researchers are currently searching for a simple animal model system to assist in finding either a cure for heart-related diseases or a novel gene capable of regulating heart development.
Franz et al. (1991, Eur. Heart Jour., Vol. 12, page 210) taught producing a transgenic mice comprising a transgene encoding the firefly luciferase gene under the control of the rat cmlc2 promoter wherein the mice specifically express luciferase in the heart. In brief, two DNA constructs containing a luciferase reporter gene (LRG) under the control of the rat cardiac myosin-light-chain-2 (MLC-2) prmoter/enhancer (P/E) were injected into fertilized mouse oocytes. The first construct, designated (2.1 kb/Luc), spanned 20.1 kb of the 5′ noncoding region of MLC-2. The second construct designated (260 bp/Luc) contained the minimal required sequence for cardiac myocyte specific expression as demonstrated in vitro. Hearts of transgenic mice expressed up to 100 pg LRG per mg of extracted protein. Neither in skeletal muscle, uterus or other organs LRG activity was detected. Hence, Franz et al. concluded that rat cardiac MLC-2 P/E is exclusively active in the myocardium, providing the possibility of gene targeting to the heart of transgenic animals.
It is noted that the in vivo model system of the transgenic mice illustrated above has some drawbacks. The mouse embryos developed from fertilized oocytes with two DNA constructs injected thereinto cannot allow an investigator to observe expression of the transgene directly. In other words, because embryos of mice are not transparent, the investigator cannot observe gene expression from early developmental stage, e.g. embryonic stage, to late developmental stage, e.g. mature stage, of mice. Under the situation, sacrifices of valuable animals, such as mice, are unavoidable.
Fish are the simplest vertebrates with heart organs. Among these fish, those which develop from transparent embryos, such as zebrafish (Danio rerio) and medaka (Oryzias latipes), have become an alternative model system for heart-related researches. The tendency derives from the following reasons: 1) organ developments can be easily observed since embryos of the fish are transparent. In this case, physiological and pathological variations, which may result from environmental pollutants, therapeutic drugs or other factors, during organogenesis can be facilely investigated as well. 2) experimental and investigative duration is greatly shortened due to rapid proceeding of embryogenesis. For example, the heart of the zebrafish starts to beat at about 24 hours postfertilization (hpf) and well develops within 48 hpf. 3) sacrifices of fish are unnecessary because contractibility of the heart can be directly observed from their appearances; and 4) large-scale screenings for mutant species are allowed.
Although using fish described above to serve as model systems possess many advantages, such model systems labeled with heart-specific fluorescence in vivo have not been established yet.